Controlling supplies for DNA and RNA synthesis

The mTORC1 protein kinase complex regulates anabolic metabolism and coordinates cellular signals that promote growth with availability of required precursor metabolites. Signaling through mTORC1 controls pyrimidine synthesis. Ben-Sahra et al. found that mTORC1 also functions by a different mechanism to regulate purine biosynthesis, thus generating precursors for the synthesis of RNA and DNA (see the Perspective by Ma and Jones). Signaling by mTORC1 caused accumulation of the transcription factor ATF4, which enhances production of the enzyme methylenetetrahydrofolate dehydrogenase 2, thus leading to increased production of the purine nucleotides needed for cell growth.

Abstract

In response to growth signals, mechanistic target of rapamycin complex 1 (mTORC1) stimulates anabolic processes underlying cell growth. We found that mTORC1 increases metabolic flux through the de novo purine synthesis pathway in various mouse and human cells, thereby influencing the nucleotide pool available for nucleic acid synthesis. mTORC1 had transcriptional effects on multiple enzymes contributing to purine synthesis, with expression of the mitochondrial tetrahydrofolate (mTHF) cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) being closely associated with mTORC1 signaling in both normal and cancer cells. MTHFD2 expression and purine synthesis were stimulated by activating transcription factor 4 (ATF4), which was activated by mTORC1 independent of its canonical induction downstream of eukaryotic initiation factor 2α eIF2α phosphorylation. Thus, mTORC1 stimulates the mTHF cycle, which contributes one-carbon units to enhance production of purine nucleotides in response to growth signals.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase integrates diverse growth signals to control nutrient-consuming biosynthetic processes, such as protein and lipid synthesis (1). mTORC1 also acutely stimulates the de novo synthesis of pyrimidine nucleotides through a posttranslational mechanism leading to increased intracellular pools of pyrimidines available for RNA and DNA synthesis (2, 3). Whether mTORC1 also influences the synthesis of purine nucleotides is unknown. Purines are enzymatically assembled on a 5-phosphoribosyl pyrophosphate (PRPP) molecule derived from the pentose phosphate pathway, with carbon and nitrogen moieties donated by nonessential amino acids and one-carbon formyl units from the tetrahydrofolate (THF) cycle (Fig. 1A).

(A) Schematic of the de novo purine synthesis pathway. (B and C) Normalized peak areas of 15N-labeled intermediates of purine (B) and pyrimidine (C) synthesis, measured by targeted LC-MS/MS, from serum-deprived Tsc2+/+ and Tsc2−/− MEFs treated with vehicle or rapamycin (20 nM) for 1 hour or 12 hours and labeled (20 min) with 15N-glutamine. (D and E) Metabolite abundance from wild-type MEFs treated as in (B) and (C) but stimulated with insulin (500 nM) for 1 hour or 16 hours. (F and G) Relative incorporation of radiolabel from 14C-glycine or 3H-adenine into RNA and DNA from serum-deprived Tsc2+/+ and Tsc2−/− MEFs treated with vehicle or rapamycin (8 hours, 20 nM) (F) or wild-type MEFs stimulated with insulin (6 hours, 100 nM) with or without rapamycin. (H) The given cells were labeled as in (F). [(B) to (H)] Data are presented as mean ± SD of biological triplicates and are representative of at least two independent experiments. *P < 0.05 by two-tailed Student’s t test.

To determine whether mTORC1 signaling affects de novo purine synthesis, we used targeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) to measure relative flux of stable isotope-labeled glutamine (amide-15N), which is incorporated into the purine ring at two positions (Fig. 1A). mTORC1 activation in response to both genetic [tuberous sclerosis complex 2 (Tsc2) null mouse embryo fibroblasts (MEFs)] and physiologic (insulin treatment of wild-type MEFs) stimuli resulted in increased amounts of doubly labeled 15N-purine intermediates, and this labeling was attenuated in cells treated for 12 to 16 hours with the mTORC1 inhibitor rapamycin (Fig. 1, B and D, and fig. S1, A to E). Unlike de novo pyrimidine synthesis, measured in the same metabolite extracts as the intermediate 15N-carbamoyl-aspartate (Fig. 1, C and E, and fig. S1C) (2, 3), a shorter 1-hour stimulation with insulin or treatment with rapamycin failed to respectively increase or decrease purine flux (Fig. 1, B and D, and fig. S1, B and D). Similar results were obtained when flux from 13C-glycine into purine intermediates was measured (fig. S1F). mTORC1 activation through either loss of Tsc2 or stimulation of cells with insulin increased flux through de novo purine synthesis into nucleic acids, as measured by 14C-glycine incorporation into RNA and DNA, without pronounced effects on the incorporation of an exogenously provided purine base (3H-adenine) (Fig. 1, F and G, and fig. S1, G to J). Likewise, rapamycin decreased 14C-glycine flux into RNA in primary mouse hepatocytes and a panel of human cell lines (Fig. 1H).

The delayed timing of the respective inhibitory and stimulatory effects of rapamycin and insulin on purine synthesis, relative to that of pyrimidine synthesis (2, 3), suggested that mTORC1 might regulate this pathway through transcriptional mechanisms. Transcripts for specific enzymes within the purine pathway or essential supporting pathways, including the pentose phosphate pathway, serine synthesis, and the THF cycle (fig. S2A), were increased in Tsc2-deficient cells and sensitive to rapamycin treatment (Fig. 2A and fig. S2, B to D). Among these genes, methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), encoding an enzyme in the mTHF cycle, showed the greatest mTORC1-dependent induction. Of those genes altered by mTORC1 signaling, Mthfd2 was among the few that also showed corresponding changes in protein abundance, which were sensitive to both rapamycin and the mTOR kinase inhibitor Torin 1 (Fig. 2B). MTHFD2 was reduced in cells treated with rapamycin for 8 hours (fig. S3A), which was also sufficient to reduce de novo purine synthesis in these cells (Fig. 1F).

Fig. 2MTHFD2 is induced downstream of mTORC1 and is required for de novo purine synthesis.

(A) Heat map of relative gene expression in serum-deprived MEFs treated 15 hours with vehicle or rapamycin (20 nM). Transcripts listed from highest to lowest fold increase in Tsc2−/− relative to Tsc2+/+ MEFs for each category. (B) Immunoblots from cells treated as in (A), but also with Torin 1 (250 nM) treatment. Biological duplicates shown. (C) MTHFD2 transcript (graphs) and protein (immunoblots) abundance in the given cell lines treated with vehicle or rapamycin (20 nM, 16 hours). (D) Schematic of serine synthesis, cytosolic and mitochondrial THF pathways, and their relation to de novo purine synthesis. (E) Normalized peak areas of 15N-labeled purine intermediates, measured by targeted LC-MS/MS, from Tsc2+/+ and Tsc2−/− MEFs 48 hours after transfection with Mthfd2 siRNAs or nontargeting controls (siCtl) and labeled (20 min) with 15N-glutamine. (F) Relative incorporation of radiolabel from 14C-serine, 14C-glycine, or 14C-formate (8-hour labeling) into RNA from Tsc2+/+ and Tsc2−/− MEFs treated as in (E). (G) Relative cell number 96 hours after transfecting Tsc2−/− MEFs as in (E), with growth in low (1%) serum with or without formate (1 mM) for final 60 hours. [(C), (E), (F), and (G)] Data are graphed as mean ± SD of biological triplicates and are representative of at least two independent experiments. *P < 0.05 by two-tailed Student’s t test.

Expression of MTHFD2 was broadly regulated by mTORC1 signaling in distinct settings. Insulin increased MTHFD2 mRNA and protein in a rapamycin-sensitive manner in wild-type MEFs (fig. S3, B and C), and these were also decreased by rapamycin in primary mouse hepatocytes and various human cancer cell lines (Fig. 2C and fig. S3D). MTHFD2 is the most highly overexpressed metabolic enzyme in human cancers (4). Our data suggest that mTORC1, which is frequently activated in cancer (5), might contribute to increased MTHFD2 expression in tumors. In 859 human breast cancer samples (6), elevated mTORC1 signaling, as scored by the abundance of phospho-S6, was associated with increased expression of MTHFD2 and other mTHF cycle genes and, to a lesser extent, enzymes of the serine synthesis pathway. mTORC1 activation did not correlate with expression of cytosolic THF cycle genes (fig. S3, E to G).

The cytosolic and mitochondrial THF cycles produce one-carbon formyl groups for various cellular processes, including de novo purine synthesis (Fig. 2D and fig. S2A) (7–11). To determine whether the mTORC1-mediated induction of MTHFD2 contributes to purine synthesis, we measured the effects of small interfering RNA (siRNA)–mediated depletion of MTHFD2 on flux from 15N-glutamine into purine intermediates. Indeed, MTHFD2 depletion lowered flux through de novo purine synthesis without affecting mTORC1 signaling (Fig. 2E and fig. S3, H and I). Formate produced by the mTHF cycle can exit the mitochondria and be converted to the one-carbon donor N10-formyl THF in the cytosol (Fig. 2D and fig. S2A). Tsc2-null cells with activated mTORC1 displayed enhanced incorporation into RNA of radiolabeled carbon from multiple substrates specific for purine synthesis, including 14C-serine, 14C-glycine, and 14C-formate. Depletion of MTHFD2 decreased the incorporation from 14C-serine and 14C-glycine but did not influence incorporation of 14C-formate (Fig. 2F and fig. S4A). These data suggest that MTHFD2 is required for the mTORC1-induced increase in purine synthesis because it increases formate production, with exogenous formate bypassing the need for this enzyme. This is further supported by the fact that MTHFD2 depletion consistently slowed proliferation in Tsc2−/− MEFs, without substantial effects on cell size, and exogenous formate attenuated this effect (Fig. 2I and fig. S4, B and C). MTHFD2 depletion also blocked 14C-glycine incorporation into RNA in five human cell lines, and, similar to mTORC1 depletion with Raptor siRNAs, loss of MTHFD2 slowed proliferation of these cells (fig. S4, D and E).

We also measured effects of candidate transcription factors downstream of mTORC1 (12–15) on incorporation of 14C-glycine into RNA and DNA. Of the six transcription factors tested, depletion of the sterol regulatory element binding proteins (SREBP1 and 2), c-Myc, or activating transcription factor 4 (ATF4) specifically attenuated the increase in de novo purine synthesis in Tsc2-deficient cells, without decreasing incorporation of 3H-adenine (Fig. 3A and fig. S5A). Of these three transcription factors, only depletion of ATF4 decreased MTHFD2 transcripts and protein in Tsc2-deficient cells (Fig. 3, B and C) and abolished MTHFD2 protein in wild-type MEFs stimulated with insulin and in human embryonic kidney 293E (HEK293E) cells (fig. S5, B and C). ATF4 depletion also decreased mRNA for another mTHF gene (Shmt2), as well as genes of the serine synthesis pathway (Psat1 and Psph), with lesser effects on their protein products (Fig. 3C and fig. S5D) (16, 17). Consistent with the role of these enzymes in de novo purine synthesis, ATF4 depletion blocked the mTORC1-induced increase in flux from 15N-glutamine into purine intermediates (fig. S5E).

Fig. 3ATF4 is required for mTORC1 to induce MTHFD2 expression and purine synthesis.

(A) Relative incorporation of radiolabel from 14C-glycine or 3H-adenine (8-hour labeling) into RNA and DNA from Tsc2+/+ and Tsc2−/− MEFs transfected with the indicated siRNAs is shown relative to that from Tsc2+/+ MEFs with control siRNAs. (B) Relative Mthfd2 transcript amounts from cells transfected as in (A). (C) Immunoblots of proteins in Tsc2−/− MEFs transfected as in (A). (D) Immunoblots from HEK293E cells expressing empty vector (Vec) or Flag-ATF4 (ATF4) treated, where indicated, with rapamycin (15 hours, 20 nM). [(C) and (D)] Biological duplicates shown. (E) MTHFD2 transcript abundance from cells transfected as in (D). *P < 0.05 by two-tailed Student’s t test. (F) Cells transfected as in (D) were subjected to ChIP with control immunoglobulin G, antibodies to Flag, or antibodies to Pol II. Bound promoter regions for the given genes were quantified and shown normalized to control IgG. (G) Relative incorporation of radiolabel from 14C-glycine or 14C-formate (8-hour labeling) into RNA from Tsc2+/+ and Tsc2−/− MEFs transfected with the indicated siRNAs is shown as in (A). [(A), (B), (E), (F), and (G)] Data are mean ± SD of biological triplicates and are representative of at least two independent experiments.

Overexpression of an ATF4 cDNA lacking its 5′-untranslated region increased the abundance of MTHFD2 mRNA and protein and rendered its expression resistant to rapamycin (Fig. 3, D and E), without effects on the cytosolic isoform, MTHFD1 (fig. S5F). Alignment of the human, mouse, and rat MTHFD2 promoters revealed a highly conserved, consensus DNA-binding motif for ATF4 (fig. S5G). Chromatin immunoprecipitation (ChIP) assays demonstrated that ATF4 bound to the MTHFD2 promoter, and ATF4 expression increased Pol II binding to this promoter (Fig. 3F). As controls, ATF4 bound the promoter of its established target CHOP but not GAPDH. Like loss of MTHFD2, depletion of ATF4 decreased flux from 14C-glycine into RNA but failed to block incorporation of 14C-formate (Fig. 3G and fig. S5H). Thus, MTHFD2 is a transcriptional target of ATF4, and ATF4 promotes de novo purine synthesis, at least in part, through its regulation of formate production by the mTHF cycle.

ATF4 DNA-binding elements are overrepresented in the promoters of mTORC1-regulated genes (12), which suggests that mTORC1 might activate ATF4. Tsc2-deficient cells had increased ATF4 that was lowered by 2 to 4 hours of rapamycin treatment (Fig. 4A). Loss of TSC2 function in mouse neurons, through conditional deletion of exon 3 (18), activated mTORC1 signaling and increased both ATF4 and its target MTHFD2 in the brain (Fig. 4B). Treatment of a panel of cancer cell lines, primary mouse hepatocytes, or HEK293E cells with rapamycin for 4 hours or less decreased ATF4 (Fig. 4C and fig. S6, A and B). The shorter-term effects of mTORC1 inhibition with rapamycin or activation with insulin did not correspond with changes in ATF4 transcripts (fig. S6, B and C), but prolonged stimulation or inhibition of mTORC1 caused transcriptional changes (fig. S6D) (16). Treatment of cells with the proteasome inhibitor MG132 increased ATF4 but did not affect its regulation by mTORC1 (Fig. 4D). Therefore, the primary mechanism of regulation of ATF4 by mTORC1 is neither through transcription nor stability, nor does it appear to influence nuclear shuttling of ATF4 (fig. S6E). The insulin-stimulated increase in ATF4 was blocked in cells upon inhibition oftranslation with cycloheximide (Fig. 4E) but wasunaffected by inhibition of transcription with actinomycin D (fig. S6F). Thus, mTORC1 appears to increase ATF4 abundance by promoting its translation.

The translation of ATF4 is well established to be regulated downstream of eukaryotic initiation factor 2α (eIF2α) phosphorylation on Ser51 in response to cellular stresses, including amino acid deprivation and endoplasmic reticulum (ER) stress (19–21). Unlike growth factor–induced ATF4, the up-regulation of ATF4 upon amino acid starvation or exposure to the ER stress–inducing agent tunicamycin was largely resistant to rapamycin (Fig. 4F). The induction of ATF4 by tunicamycin was abolished in a Ser51Ala knock-in mutant of eIF2α (A/A) (21), but the insulin-stimulated, rapamycin-sensitive increase in ATF4 was similar between these cells and their wild-type (S/S) counterparts (Fig. 4G). These data reveal a regulatory input from mTORC1 to ATF4 that is independent of its established mechanism of regulation by cellular stress.

This study adds purine synthesis to the list of anabolic processes induced by mTORC1 in both normal and cancer cells (1). In response to growth signals, mTORC1 activates ATF4, which stimulates expression of MTHFD2 and other enzymes of the serine synthesis and mTHF cycle, thereby increasing production of formyl units required for de novo purine synthesis (Fig. 4H).

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